My colleagues say that my transfection efficiency is low because I might have mycoplasma contamination. is this true?

I use a control pEGFP and the transfection efficiency is above 80% and easily detectable by western blot.

But the cloned pEGFP which has a viral protein right before the GFP tag has a very low efficiency and not detectable by wb.

-Curtis-

There could be all sorts of explanations for the inefficiency of the transfection. I would suspect that the protein is not well expressed from the plasmid due to size of product, toxicicty, or incorrect sequence before I suspected mycoplasma.

-bob1-

thanks bob1,
but what are the common reasons for not expressing? one of my plasmids must express well, because there are many papers published with it. it is pcDNA3-FLAG-BimL.

-Curtis-

Have you sequenced it to determine if the insert is correct and in-frame? You could have been sent a batch with incorrect sequence at some point, or it could have mutated as sometimes happens.

-bob1-

yes, it is 100% match, with no gaps

-Curtis-

Plasmids transfect most effectively when supercoiled, if you are using a mini-prep or other alkaline lysis based preparation (most kits are) then if you leave the plasmids in the alkaline step for too long, you end up with nicked or linear plasmids, which apparently don't transfect so well. Run your plasmids out on a gel and have a look for the supercoiled band.

-bob1-

Thanks so much,

I use conventional method for plasmid preparation (solution I, II, III, phenol/chloroform). I'll have a look at supercoiled then.

-Curtis-

Hi Curtis~
The fact that your control plasmid transfects/expresses without any problems would indicate that the problem is not with your cells or transfection but rather it is specific to this DNA. Either the prep itself is a problem or the expression of this specific gene is toxic. Do you see a loss of cells? If you transfect the plate at say 80% confluence, the next day do you still have 80% or do you see a bunch of floating cells? Do you have an inverted microscope that you are using to check for the GFP? What is the absorbance reading of the DNA prep at 230, 260 and 280? How long do you wait after transfection before harvesting the cells? I work with a domain of a gene that is extremely toxic and I can only let the cells go a maximum of 20 hours before they almost completely die. You might might want to check your cells for expression at earlier time points.

-rkay447-

Perhaps endotoxin contamination?

If the plasmid was harvested from cells that have been growing a little too long , the contamination of endotoxins might be high enough to get through the phenol extraction clean up procedure. You could try several more phenol -extraction steps or better yet pass your DNA through an endotoxin removal kit.

If you don't have such a kit, passing your plasmid DNA through Qiagen gel extraction column (add 5x volume QG buffer to DNA sample) will remove most endotoxin contamination. (use two washes)

-perneseblue-

thanks guys,
my viral gene has a BH3 domain that is similar to Bax pro-apoptotic proteins and I have a lot of dead cells 16hrs post infection. my boss also says that this could be the reason i'm not seeing much efficiency. because the moment the gene is expressed it'll trigger apoptosis.

endotoxin could be another problem to consider. but i usually incubate 16-18hrs and also prepared fresh phenol/chloroform! so why is this happening?